The polarized growth and division of budding yeast cells depend on the precise spatial and temporal regulation of actin filament assembly at cell cortex. Determining the mechanism of this regulation is important for understanding many eukaryotic morphogenetic processes. Our long term goal is to identify the gene products that directly control actin polymerization in yeast and understand the biochemical basis of their activities and how the activities are controlled by signaling pathways that govern yeast cell polarity development. We will achieve this goal by combining the powerful yeast genetic techniques with biochemical purification and in vitro reconstitution. Studies proposed in this grant are focused on two critical components of actin assembly sites at the bud cortex of yeast cells. The first component is Arp2/3 complex, a multi- subunit protein complex that is conserved in many eukaryotic organisms. We have purified the yeast Arp2/3 complex and identified all of the subunits. We will carry out genetic and biochemical analyses to determine the role of Arp2/3 complex in cortical actin filament assembly. The second cytoskeletal component to be studied is Bee1, a yeast homolog of the Wiskott-Aldrich syndrome protein. We have obtained strong in vivo and in vitro evidence indicating that Bee1 protein is a key functional component of cortical actin assembly sites. We will determine how Bee1 protein and Arp2/3 complex acts synergistically in the assembly of cortical actin filaments. Our findings should be significant for understanding similar processes in mammalian organisms because of the conservation in actin cytoskeletal components. The analysis of Bee1 may shed light on the decease mechanism of Wiskott-Aldrich syndrome, a severe immunodeficiency that correlates with defects in blood cell morphogenesis.